Adeno-associated virus (AAV) may be considered as one of the most promising viral vectors for human gene therapy. AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is present in many humans it has never been associated with any disease. In view of these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases.
Host cells that sustain AAV replication in vitro are all derived from mammalian cell types. Therefore, rAAV for use in gene therapy has thus far mainly been produced on mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines (see e.g. U.S. Pat. No. 6,156,303, U.S. Pat. No. 5,387,484, U.S. Pat. No. 5,741,683, U.S. Pat. No. 5,691,176, U.S. Pat. No. 5,688,676, US 20020081721, WO 00/47757, WO 00/24916, and WO 96/17947). rAAV vectors are typically produced in such mammalian cell culture systems by providing DNA plasmids that contain the therapeutic gene flanked by the origin of AAV replication (inverted terminal repeats or ITRs), genes for AAV replication proteins Rep78, Rep68, Rep52, and Rep40, and genes for virion or structural proteins VP1, VP2, and VP3. In addition, a plasmid containing early genes from adenovirus (E2A, E4ORF6, VARNA) is provided to enhance the expression of the AAV genes and improve vector yield (see e.g. Grimm et al., 1998, Hum. Gene Ther. 9: 2745-2760). However, in most of these mammalian cell culture systems, the number of AAV particles generated per cell is on the order of 104 particles (reviewed in Clark, 2002, Kidney Int. 61(Suppl. 1): 9-15). For a clinical study, more than 1015 particles of rAAV may be required. To produce this number of rAAV particles, transfection and culture with approximately 1011 cultured human 293 cells, the equivalent of 5,000 175-cm2 flasks of cells, would be required, which means transfecting up to 1011 293 cells. Therefore, large scale production of rAAV using mammalian cell culture systems to obtain material for clinical trials has already proven to be problematic, production at commercial scale may not even be feasible. Furthermore there is always the risk, that a vector for clinical use that is produced in a mammalian cell culture will be contaminated with undesirable, perhaps pathogenic, material present in the mammalian host cell.
To overcome these problems of mammalian productions systems, recently, an AAV production system has been developed using insect cells (Urabe et al., 2002, Hum. Gene Ther. 13: 1935-1943; US 20030148506 and US 20040197895). For production of AAV in insect cells from the baculovirus expression system some modifications were necessary because in mammalian cells expression of the three AAV capsid proteins (VP1, VP2 and VP3) in the correct stoichiometry relies on a combination of alternate usage of two splice acceptor sites and the suboptimal utilization of a ACG initiation codon for VP2 that will not be accurately reproduced by insect cells. Correct stoichiometry of the three capsid proteins is important for infectivity of the AAV particles. It is known that AAV particles containing reduced amounts of VP1 are less infectious and that VP1 contains phospholipase A2 activity which has a function in infectivity (Girod et al., 2002 J. Gen. Virol. 83: 973-8).
Therefore, for the expression of the three capsid proteins Urabe et al. (2002, supra) use a construct that is transcribed into a single polycistronic messenger that is able to express all three VP proteins without requiring splicing. To aim for production of the three capsid proteins in the correct stoichiometry, the VP1 reading frame, the first initiator codon that is seen by the scanning ribosome, has been endowed with the suboptimal initiator codon ACG and sequences surrounding this codon have been optimized. Urabe et al. (2002, supra) report that ribosome scanning through in insects cells leads to a stoichiometry of the three viral capsid proteins that is close to wild-type AAV.
The present inventors have, however, found that in AAV vectors produced in the baculovirus system VP1 is still expressed at a suboptimal level relative to VP2 and that this results in reduced infectivity in in vitro and in vivo studies in mice as compared to e.g. conventional AAV vectors produced on mammalian 293 cells. Hence, there is still a need for a baculovirus-based production system for rAAV vectors with improved infectivity.